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Expressed sequence tags for bovine muscle satellite cells, myotube formed-cells and adipocyte-like cells.

Lee EJ, Kamli MR, Pokharel S, Malik A, Tareq KM, Roouf Bhat A, Park HB, Lee YS, Kim S, Yang B, Chung KY, Choi I - PLoS ONE (2013)

Bottom Line: DDAH2 was up-regulated during myognesis and knockdown of DDAH2 by siRNA significantly decreased myogenin (MYOG) expression corresponding with the slight change in cell morphology.In contrast, HBA2 was up-regulated during ALC formation and resulted in decreased intracellular lipid accumulation and CD36 mRNA expression upon knockdown assay.Overall, the collection of ESTs generated in this study provides a starting point for the identification of novel genes involved in MFC and ALC formation, which in turn offers a fundamental resource to enable better understanding of the mechanism of muscle differentiation and transdifferentiation.

View Article: PubMed Central - PubMed

Affiliation: School of Biotechnology, Yeungnam University, Gyeongsan, Republic of Korea ; Bovine Genome Resources Bank, Yeungnam University, Gyeongsan, Republic of Korea.

ABSTRACT

Background: Muscle satellite cells (MSCs) represent a devoted stem cell population that is responsible for postnatal muscle growth and skeletal muscle regeneration. An important characteristic of MSCs is that they encompass multi potential mesenchymal stem cell activity and are able to differentiate into myocytes and adipocytes. To achieve a global view of the genes differentially expressed in MSCs, myotube formed-cells (MFCs) and adipocyte-like cells (ALCs), we performed large-scale EST sequencing of normalized cDNA libraries developed from bovine MSCs.

Results: A total of 24,192 clones were assembled into 3,333 clusters, 5,517 singletons and 3,842contigs. Functional annotation of these unigenes revealed that a large portion of the differentially expressed genes are involved in cellular and signaling processes. Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis of three subsets of highly expressed gene lists (MSC233, MFC258, and ALC248) highlighted some common and unique biological processes among MSC, MFC and ALC. Additionally, genes that may be specific to MSC, MFC and ALC are reported here, and the role of dimethylarginine dimethylaminohydrolase2 (DDAH2) during myogenesis and hemoglobin subunit alpha2 (HBA2) during transdifferentiation in C2C12 were assayed as a case study. DDAH2 was up-regulated during myognesis and knockdown of DDAH2 by siRNA significantly decreased myogenin (MYOG) expression corresponding with the slight change in cell morphology. In contrast, HBA2 was up-regulated during ALC formation and resulted in decreased intracellular lipid accumulation and CD36 mRNA expression upon knockdown assay.

Conclusion: In this study, a large number of EST sequences were generated from the MSC, MFC and ALC. Overall, the collection of ESTs generated in this study provides a starting point for the identification of novel genes involved in MFC and ALC formation, which in turn offers a fundamental resource to enable better understanding of the mechanism of muscle differentiation and transdifferentiation.

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DDAH2 knockdown in C2C12 cells during myogenesis.mRNA expression of DDAH2 and MYOG after DDAH2kd during differentiation in C2C12 at Day 2 (A). Representative cell picture showing morphological changes in DDAH2kd cells (B). Mock represents control (mean ± S.D., n= 3). p-value indicates the statistical significance of the data and different letters show significant differences among groups.
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pone-0079780-g005: DDAH2 knockdown in C2C12 cells during myogenesis.mRNA expression of DDAH2 and MYOG after DDAH2kd during differentiation in C2C12 at Day 2 (A). Representative cell picture showing morphological changes in DDAH2kd cells (B). Mock represents control (mean ± S.D., n= 3). p-value indicates the statistical significance of the data and different letters show significant differences among groups.

Mentions: To confirm the role of genes identified during differentiation and transdifferentiation, gene knockdown by siRNA was employed in DDAH2and HBA2. DDAH2kd by siRNA showed a 40% reduction in mRNA expression with a slight change in cell morphology (Figure 5A&B). Interestingly, DDAH2kd showed a decrease in MYOG upto 50% during myogenesis (Figure 5A). Furthermore, HBA2 silencing in C2C12 cells showed an upto 70% reduction in its expression during transdifferentiation (Figure 6A). CD36, which is a marker gene for adipogenesis, was checked in HBA2kd and its expression was found to be reduced up to 50%, whereas FABP4 was slightly decreased (Figure 6 B). The effects of HBA2kd on intracellular lipid formation followed by its treatment with transdifferentiation media were also evaluated at day 5 by Oil Red O (O-R-O) staining and the results showed a 20% reduction in intracellular fat during ALC formation (Figure 6C& D).


Expressed sequence tags for bovine muscle satellite cells, myotube formed-cells and adipocyte-like cells.

Lee EJ, Kamli MR, Pokharel S, Malik A, Tareq KM, Roouf Bhat A, Park HB, Lee YS, Kim S, Yang B, Chung KY, Choi I - PLoS ONE (2013)

DDAH2 knockdown in C2C12 cells during myogenesis.mRNA expression of DDAH2 and MYOG after DDAH2kd during differentiation in C2C12 at Day 2 (A). Representative cell picture showing morphological changes in DDAH2kd cells (B). Mock represents control (mean ± S.D., n= 3). p-value indicates the statistical significance of the data and different letters show significant differences among groups.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3818215&req=5

pone-0079780-g005: DDAH2 knockdown in C2C12 cells during myogenesis.mRNA expression of DDAH2 and MYOG after DDAH2kd during differentiation in C2C12 at Day 2 (A). Representative cell picture showing morphological changes in DDAH2kd cells (B). Mock represents control (mean ± S.D., n= 3). p-value indicates the statistical significance of the data and different letters show significant differences among groups.
Mentions: To confirm the role of genes identified during differentiation and transdifferentiation, gene knockdown by siRNA was employed in DDAH2and HBA2. DDAH2kd by siRNA showed a 40% reduction in mRNA expression with a slight change in cell morphology (Figure 5A&B). Interestingly, DDAH2kd showed a decrease in MYOG upto 50% during myogenesis (Figure 5A). Furthermore, HBA2 silencing in C2C12 cells showed an upto 70% reduction in its expression during transdifferentiation (Figure 6A). CD36, which is a marker gene for adipogenesis, was checked in HBA2kd and its expression was found to be reduced up to 50%, whereas FABP4 was slightly decreased (Figure 6 B). The effects of HBA2kd on intracellular lipid formation followed by its treatment with transdifferentiation media were also evaluated at day 5 by Oil Red O (O-R-O) staining and the results showed a 20% reduction in intracellular fat during ALC formation (Figure 6C& D).

Bottom Line: DDAH2 was up-regulated during myognesis and knockdown of DDAH2 by siRNA significantly decreased myogenin (MYOG) expression corresponding with the slight change in cell morphology.In contrast, HBA2 was up-regulated during ALC formation and resulted in decreased intracellular lipid accumulation and CD36 mRNA expression upon knockdown assay.Overall, the collection of ESTs generated in this study provides a starting point for the identification of novel genes involved in MFC and ALC formation, which in turn offers a fundamental resource to enable better understanding of the mechanism of muscle differentiation and transdifferentiation.

View Article: PubMed Central - PubMed

Affiliation: School of Biotechnology, Yeungnam University, Gyeongsan, Republic of Korea ; Bovine Genome Resources Bank, Yeungnam University, Gyeongsan, Republic of Korea.

ABSTRACT

Background: Muscle satellite cells (MSCs) represent a devoted stem cell population that is responsible for postnatal muscle growth and skeletal muscle regeneration. An important characteristic of MSCs is that they encompass multi potential mesenchymal stem cell activity and are able to differentiate into myocytes and adipocytes. To achieve a global view of the genes differentially expressed in MSCs, myotube formed-cells (MFCs) and adipocyte-like cells (ALCs), we performed large-scale EST sequencing of normalized cDNA libraries developed from bovine MSCs.

Results: A total of 24,192 clones were assembled into 3,333 clusters, 5,517 singletons and 3,842contigs. Functional annotation of these unigenes revealed that a large portion of the differentially expressed genes are involved in cellular and signaling processes. Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis of three subsets of highly expressed gene lists (MSC233, MFC258, and ALC248) highlighted some common and unique biological processes among MSC, MFC and ALC. Additionally, genes that may be specific to MSC, MFC and ALC are reported here, and the role of dimethylarginine dimethylaminohydrolase2 (DDAH2) during myogenesis and hemoglobin subunit alpha2 (HBA2) during transdifferentiation in C2C12 were assayed as a case study. DDAH2 was up-regulated during myognesis and knockdown of DDAH2 by siRNA significantly decreased myogenin (MYOG) expression corresponding with the slight change in cell morphology. In contrast, HBA2 was up-regulated during ALC formation and resulted in decreased intracellular lipid accumulation and CD36 mRNA expression upon knockdown assay.

Conclusion: In this study, a large number of EST sequences were generated from the MSC, MFC and ALC. Overall, the collection of ESTs generated in this study provides a starting point for the identification of novel genes involved in MFC and ALC formation, which in turn offers a fundamental resource to enable better understanding of the mechanism of muscle differentiation and transdifferentiation.

Show MeSH
Related in: MedlinePlus